Stabilized thrombin

ABSTRACT

The present invention is directed to compounds, methods for stabilizing thrombin activity with a thrombin binding oligonucleotide and to stabilized thrombin. The thrombin binding oligonucleotide is capable of inhibiting thrombin activity whereby the inhibition can be reversed with an antisense oligonucleotide.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which is submittedconcomitantly with this application via EFS-Web in ASCII format and ishereby incorporated by reference in its entirety. Said ASCII copy,created on Aug. 11, 2014 is named “Sequence Listing” and is 3 kilobytesin size.

FIELD OF THE INVENTION

Provided herein are compounds, compositions comprising same and methodsuseful for reversibly stabilizing thrombin activity and extendingthrombin's shelf-life. In particular, disclosed herein are reversiblethrombin binding oligonucleotides capable of interacting with thrombinand inhibiting thrombin activity and an antisense oligonucleotidethereto, and compositions and methods of use therefore to stabilizethrombin activity in a liquid thrombin formulation.

BACKGROUND

Thrombin is a serine protease which serves as an active component inseveral hemostasis products. For example, fibrin sealants typicallycomprise a fibrinogen component and a thrombin component. When bothcomponents are mixed (e.g. when applied to a bleeding wound or surgicalincision) thrombin cleaves fibrinogen and a fibrin polymer is formed.Concentrated purified thrombin in liquid form displays a reduction inactivity during prolonged storage, primarily as a result of autolysis.

Hemostatic formulations containing liquid thrombin have special handlingrequirements in order to maintain thrombin's biologic activity andprevent autolytic degradation. For example, liquid thrombin requiresfreezing or the addition of protease inhibitors to maintain shelf-lifestability. The shortcoming of the liquid thrombin solutions in use todayare manifold: In the clinic and operating room, freezing is expensiveand not always feasible, and promiscuous protease inhibitors mayadversely affect activity of thrombin and other proteases in thehemostasis pathway once the fibrin sealant or the thrombin is applied.

Liquid thrombin preparations may be made into a lyophilized medicalpreparation, which is used after dissolving at the time of use. However,liquid preparations are advantageous compared with the lyophilizedpreparations in that they can be easily administered without theadditional step of dissolving in a solvent prior to use. Also, thelyophilization step is costly and time consuming and may result in lossof yield.

Known compositions and methods for stabilizing thrombin areunsatisfactory and include the following: inclusion of variousnon-specific components (e.g. bulk carrier proteins such as albumins,different stabilizing sugars, general protease inhibitors etc.);formulation of the thrombin with inhibitors of thrombin activity, whichalthough may be efficient, also inactivate or inhibit the thrombin,thereby reducing its effectiveness; and formulation of a low thrombinconcentration solution, thereby necessitating administration of largeramounts of the formulation.

Several publications relate to stabilization of thrombin: for exampleInternational Patent Application Publication No. WO2008157304; U.S. Pat.Nos. 4,409,334; 7,351,561 and 8,394,372; U.S. Patent Application No.20080311104; and European Patent Nos. EP0277096 B1 and EP 0478827 B1.

There remains a need for thrombin specific compounds useful to stabilizethrombin from autolytic degradation. Preferably, the compounds arereversible thrombin inhibitors which may be used with a concentratedliquid thrombin formulation.

SUMMARY OF THE INVENTION

Provided herein is a method for the use of thrombin bindingoligonucleotides that have the exceptional ability to reversibly inhibitthrombin and stabilize its activity in an aqueous solution.

The thrombin binding oligonucleotides are such that they can bind tothrombin in a thrombin aqueous solution and inhibit, at least partially,the activity of thrombin. This inhibition of the thrombin activity bythe thrombin binding oligonucleotide can be reversed by contacting thethrombin solution comprising the thrombin binding oligonucleotide withan antisense oligonucleotide. Thus, an aqueous thrombin solution can bestabilized by contacting it with the thrombin binding oligonucleotide inthe absence of the antisense oligonucleotide. When the use of thrombinis desired, the activity of thrombin can be restored by contacting thestabilized thrombin solution with the antisense oligonucleotide. Thereversibility of the binding of the thrombin binding oligonucleotide tothrombin by adding an antisense oligonucleotide provides an unequivocaladvantage in the clinic and precludes the need to freeze and thaw aliquid thrombin solution or to reconstitute a lyophilized thrombincomponent prior to use.

The present inventors have shown for the first time that thrombinbinding oligonucleotides are capable of reversibly inhibiting andstabilizing activity of liquid thrombin and that such molecules areuseful in extending the shelf-life of thrombin.

Without wishing to be bound to theory, the thrombin binding moleculesbind thrombin to inhibit, fully or partially, thrombin autolysis. Oncethe thrombin-binding oligonucleotide bound to thrombin comes in contactwith an antisense oligonucleotide, the thrombin is released frominhibition and is capable of cleaving its heterologous substrates,including fibrinogen. The methods are further beneficial in that theyare easily carried out in the clinic.

In one aspect, provided herein is a method for stabilizing thrombinactivity in a solution (e.g. aqueous solution), the method includesinhibiting thrombin activity by contacting the thrombin with a thrombinbinding oligonucleotide, wherein the thrombin binding oligonucleotide iscapable of binding a second oligonucleotide; and wherein the inhibitionof thrombin activity can be reversed by contacting the thrombin bindingoligonucleotide with the second oligonucleotide. In some embodiments,the second oligonucleotide is an antisense oligonucleotide to thethrombin binding oligonucleotide.

In one aspect, provided herein is a method for stabilizing thrombinactivity in a solution, the method includes inhibiting thrombin activityby contacting the thrombin with a thrombin binding oligonucleotide,wherein the thrombin binding oligonucleotide is capable of binding anantisense oligonucleotide; and wherein the inhibition of thrombinactivity can be reversed by contacting the thrombin bindingoligonucleotide bound to thrombin with the antisense oligonucleotide.

In some embodiments of the method, the solution comprises a thrombinconcentration equal to or higher than 4 IU/ml and up to 15,000 IU/mlthrombin. In some embodiments, the thrombin concentration in solution is10 IU/ml-1,000 IU/ml; 20 IU/ml-15,000 IU/ml; 100 IU/ml-5,000 IU/ml; 200IU/ml-1000 IU/ml or 300 IU/ml-1000 IU/ml.

The thrombin binding oligonucleotide is selected to bind to any regionof the thrombin to which binding would inhibit thrombin activity in areversible manner; for example to inhibit thrombin activity fromautolysis. In some embodiments, the thrombin binding oligonucleotidebinds to exosite I or exosite II of thrombin or to both. In specificembodiments of the method, the thrombin binding oligonucleotide binds toexosite I. In some embodiments, the thrombin binding oligonucleotide isan aptamer comprising a DNA and/or RNA nucleotide sequence of about 10to about 60 nucleotides in length, or about 12 to about 40 nucleotidesin length, about 14 to about 35 nucleotides in length, or about 14 toabout 25 nucleotides in length. The aptamer may bind to a consecutivelinear sequence of amino acids in thrombin or may bind to a threedimensional region of thrombin. In some embodiments, the thrombinbinding aptamer is a DNA oligonucleotide comprising a nucleotidesequence set forth in SEQ ID NO:1 5′ GGTTGGTGTGGTTGG 3′, a variant ofSEQ ID NO:1 comprising a nucleotide sequence set forth in SEQ ID NO:2 5′GGGTTGGGTGTGGGTTGGG 3′ or a DNA oligonucleotide comprising a nucleotidesequence set forth in SEQ ID NO:3 5′ AGTCCGTGGTAGGGCAGGTTGGGGTGACT 3′.In specific embodiments, the nucleotide sequence of the thrombin bindingaptamer is set forth in SEQ ID NO:1. In some embodiments, the nucleotidesequence of the thrombin binding aptamer is set forth in SEQ ID NO:2. Insome embodiments, the nucleotide sequence of the thrombin bindingaptamer is set forth in SEQ ID NO:3.

In some embodiments, the thrombin binding aptamer is an RNAoligonucleotide comprising a nucleotide sequence set forth in SEQ IDNO:4 5′ GGUUGGUGUGGUUGG 3′, a variant of SEQ ID NO: 4 comprising anucleotide sequence set forth in SEQ ID NO:5 5′GGGUUGGGUGUGGGUUGGG 3′ oran RNA oligonucleotide comprising a nucleotide sequence set forth in SEQID NO:6 5′ AGUCCGUGGUAGGGCAGGUUGGGGUGACU 3′.

The antisense oligonucleotide is selected to bind to the thrombinbinding oligonucleotide. The antisense oligonucleotide includes anucleotide sequence that binds to the thrombin binding oligonucleotideand may bind to a consecutive linear sequence or to a three dimensionalstructure of the thrombin binding oligonucleotide. In some embodimentsof the method, the antisense oligonucleotide comprises a DNA and/or RNAnucleotide sequence of about 8 to about 60 nucleotides in length, orabout 10 to about 40 nucleotides in length, about 12 to about 35nucleotides in length, or about 12 to about 25 nucleotides in length.The antisense oligonucleotide may bind part of or the entire thrombinbinding oligonucleotide. In some embodiments, the antisenseoligonucleotide is a DNA oligonucleotide having a nucleotide sequenceset forth in SEQ ID NO:7 5′ CCAACCACACCAACC 3′, SEQ ID NO:8 5′CCCAACCCACACCCAACCC, or SEQ ID NO:9 5′ AGTCACCCCAACCTGCCCTACCACGGACT 3′.In some embodiments, the antisense oligonucleotide is an RNAoligonucleotide having a nucleotide sequence set forth in SEQ ID NO:105′ CCAACCACACCAACC 3′, SEQ ID NO:11 5′ CCCAACCCACACCCAACCC 3′ or SEQ IDNO:12 5′ AGUCACCCCAACCUGCCCUACCACGGACU 3′.

An antisense oligonucleotide can further be covalently or non-covalentlyattached to or associated with a molecule which may include one or moreof a nucleotide or non-nucleotide moiety, e.g. a nucleotide, anucleotide analog, an amino acid, a peptide, a polypeptide, a lipidmoiety, a carbohydrate moiety, a marker, a matrix, beads or a tag,directly or using a linker

In some embodiments of the method, the solution comprises a molar ratioof less than about 10:1 to about 1:1 thrombin bindingoligonucleotide:thrombin. In some embodiments, the ratio of the thrombinbinding oligonucleotide:thrombin in the solution is about 9:1, or about8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about3.5:1, or about 3:1, or about 2.5:1; or about 2:1 or about 1:1.

In some embodiments of the method, dissociation constant (Kd) betweenthe thrombin binding oligonucleotide and the antisense oligonucleotidein solution is equal to or less than 0.2 μM (microM).

In another aspect, provided herein is a thrombin formulation for use inapplications requiring conversion of fibrinogen to fibrin, theformulation comprising: thrombin and a thrombin binding oligonucleotide,wherein the thrombin binding oligonucleotide inhibits thrombin activityand is capable of binding an antisense oligonucleotide and wherein theinhibition of thrombin activity can be reversed by contacting theformulation with the antisense oligonucleotide.

In some embodiments of the formulation, thrombin is present in theformulation at a concentration equal to or higher than 4 IU/ml and up to15,000 IU/ml thrombin. In some embodiments, the thrombin concentrationin solution is 10 IU/ml-1,000 IU/ml; 20 IU/ml-15,000 IU/ml; 100IU/ml-5,000 IU/ml; 200 IU/ml-1000 IU/ml or 300 IU/ml-1000 IU/ml.

The thrombin binding oligonucleotide is selected to bind to any regionof the thrombin to which binding would reversibly inhibit and stabilizethrombin activity; for example fully or partially inhibit thrombinactivity from autolysis. In some embodiments of the formulation, thethrombin binding oligonucleotide binds to exosite I or exosite II ofthrombin. In specific embodiments, the thrombin binding oligonucleotidebinds to exosite I. In some embodiments, the thrombin bindingoligonucleotide is an aptamer comprising a nucleic acid sequence setforth in any of SEQ ID NOS:1-6. In one embodiment the thrombin bindingoligonucleotide is an aptamer comprising a nucleic acid sequence setforth in SEQ ID NO:1.

In some embodiments of the formulation, the thrombin bindingoligonucleotide and thrombin are present at a molar ratio of less thanabout 10:1 to about 1:1 thrombin binding oligonucleotide:thrombin. Insome embodiments, the ratio of the thrombin bindingoligonucleotide:thrombin in the formulation is about 9:1, or about 8:1,or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3.5:1,or about 3:1, or about 2.5:1; or about 2:1 or about 1:1.

In some embodiments of the formulation, the dissociation constant (Kd)between the thrombin binding oligonucleotide and the antisenseoligonucleotide in solution is equal to or less than 0.2 μM (microM).

In yet another aspect, provided herein is a kit comprising a containercomprising thrombin and a thrombin binding oligonucleotide; a containercomprising an antisense oligonucleotide to the thrombin bindingoligonucleotide; and optionally instructions for use.

The containers can be a monolithic piece including at least two chambersseparated by a septum.

In one embodiment, the chambers are divided by a septum, which is atleast partially breakable, braking the septum allows mixing theoligonucleotide inhibited thrombin and the antisense oligonucleotide.

In some embodiments, the container and/or chambers are flexible andbreaking the septum can be achieved by applying pressure (e.g. manualpressure) onto the container and/or chambers.

The size of each chamber and fill volumes are dependent e.g. on theintended use, suitable concentration ratios between the antisenseoligonucleotides, and the thrombin binding oligonucelotide, and/ordesired volume.

The container and/or chamber can comprise an opening e.g. including Maleor Female Luer Lock. The opening can be resealable.

The antisense oligonucleotide can be in powder or liquid form. The kitcan contain a liquid for reconstitution.

The kit can comprise a multi- e.g. dual chamber prefilled syringe, onechamber containing thrombin and a thrombin binding oligonucleotide andthe other chamber containing an antisense oligonucleotide e.g. a dualchamber as described in PCT Patent Publication No. WO 97/46202A1.

In some embodiments of the kit, the thrombin binding oligonucleotide iscapable of binding the antisense oligonucleotide and the binding of thethrombin binding oligonucleotide and the thrombin is reversed bycontacting the thrombin binding oligonucleotide with the antisenseoligonucleotide. In some embodiments, the antisense oligonucleotide isin solution. In alternative embodiments, the antisense oligonucleotideis solid phase or is linked to a solid phase. The kit may furthercomprise a container comprising fibrinogen. In various embodiments ofthe kit, the antisense oligonucleotide is excluded from the fibrinogencomponent.

In some embodiments of the kit, the thrombin binding oligonucleotide andthrombin are present at a molar ratio of less than about 10:1 to about1:1 thrombin binding oligonucleotide:thrombin. In some embodiments, theratio of the thrombin binding oligonucleotide:thrombin in theformulation is about 9:1, or about 8:1, or about 7:1, or about 6:1, orabout 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about 2.5:1;or about 2:1 or about 1:1.

Within one embodiment, the containers are sealed containers having alabel affixed to an exterior surface thereof. In some embodiments, theformulation is prepared for use as a fibrin sealant component. The kitmay further comprise one or more devices for the delivery of thethrombin-thrombin binding oligonucleotide; antisense oligonucleotideand/or the fibrinogen components.

In another aspect, provided is a method for converting fibrinogen tofibrin comprising:

mixing a formulation comprising: thrombin and a thrombin bindingoligonucleotide, wherein the thrombin binding oligonucleotide is capableof inhibiting thrombin activity and of binding an antisenseoligonucleotide, and wherein the inhibition of thrombin activity isreversed by the addition of the antisense oligonucleotide; the antisenseoligonucleotide and fibrinogen.

In some embodiments of the method, the ratio between the antisenseoligonucleotide and the thrombin binding oligonucleotide is in the rangeof about 1:1 to 2:1.

In some embodiments of the method, the thrombin binding oligonucleotideand thrombin are present at a molar ratio of less than about 10:1 toabout 1:1 thrombin binding oligonucleotide:thrombin. In someembodiments, the ratio of the thrombin binding oligonucleotide:thrombinin the formulation is about 9:1, or about 8:1, or about 7:1, or about6:1, or about 5:1, or about 4:1, or about 3.5:1, or about 3:1, or about2.5:1; or about 2:1 or about 1:1. Fibrinogen component can be preparedas described in the art, for example, in PCT Patent Publication No. WO93/05822 or as in the fibrin kit described in the European pharmacopeia.In some embodiments, the fibrinogen component is free of or is depletedof plasmin(ogen) as disclosed in U.S. Pat. No. 7641918 or in PCT PatentPublication No. WO 02/095019.

In some embodiments of the method, the nucleic acid sequence of thethrombin binding oligonucleotide is set forth in any of SEQ ID NOS:1-6,preferably SEQ ID NO:1.

In yet another aspect, provided herein is an antisense oligonucleotideof a thrombin binding oligonucleotide for use in reversing a bindingbetween the thrombin binding oligonucleotide and thrombin inapplications requiring converting fibrinogen to fibrin.

In some embodiments, the antisense oligonucleotide comprises any of SEQID NOS:7-12. Further provided is a composition comprising the antisenseoligonucleotide disclosed herein; and a pharmaceutically acceptablecarrier.

In another aspect, provided herein is a method of screening foroligonucleotides capable of reversibly binding and stabilizing thrombinactivity and of binding an antisense oligonucleotide. Accordingly,provided is a method for screening for an oligonucleotide capable ofreversibly binding and stabilizing the activity of thrombin in anaqueous liquid thrombin formulation, comprising

-   -   a. contacting thrombin or a fragment thereof, each exhibiting an        initial activity of 4 to 15,000 IU/ml, with a set of test        thrombin binding oligonucleotides; and identifying one or more        thrombin binding oligonucleotides which inhibit, at least        partially, the initial activity; and    -   b. contacting the thrombin bound oligonucleotide of step a) with        a set of test antisense oligonucleotides;        whereby restoration of at least 4 IU/ml thrombin activity        following step (b) indicates 1) a potential thrombin binding        oligonucleotide for thrombin stabilization and 2) a potential        antisense oligonucleotide to reverse the inhibitory effect of        the thrombin binding oligonucleotide.

In some embodiments of the screening method, the thrombin has an initialactivity of 4 to 15,000 IU/ml, about 20 IU/ml to 15,000 IU/ml, or 100IU/ml to 5,000 IU/ml, 200 IU/ml to about 1000 IU/ml or about 300 IU/mlto about 1000 IU/ml.

In some embodiments the method provides an antisense oligonucleotidethat restores thrombin activity to at least 4 IU/ml, at least about 20IU/ml, at least about 100 IU/ml or at least about 300 IU/ml, at leastabout 1000 IU/ml and up to 1500 IU/ml of the initial activity ofthrombin.

In an alternative, provided is a method for screening for anoligonucleotide capable of reversibly binding and stabilizing theactivity of thrombin in an aqueous liquid thrombin formulation,comprising

-   -   a. contacting thrombin or a fragment thereof, each exhibiting an        initial activity, with a set of test thrombin binding        oligonucleotides; and identifying one or more thrombin binding        oligonucleotides which inhibit, at least 60% of the initial        activity; and    -   b. contacting a thrombin bound oligonucleotide of step a) with a        set of test antisense oligonucleotides;        whereby restoration of more than 40% of the initial activity        following step (b) indicates 1) a potential thrombin binding        oligonucleotide for thrombin stabilization and 2) a potential        antisense oligonucleotide to reverse the inhibitory effect of        the thrombin binding oligonucleotide.

In some embodiments, the method provides stabilizing thrombin activityand restoring at least, 40%, 50% and up to 100% of the initial activityof thrombin.

In another aspect, provided herein is an antisense oligonucleotide of athrombin binding oligonucleotide for use in reversing thrombininhibition by thrombin binding oligonucleotide in applications requiringconverting fibrinogen to fibrin.

In some embodiments, the antisense oligonucleotide is bound to a solidphase.

In another aspect, provided herein is a method for reversing anoligonucleotide inhibited thrombin, the method comprising the steps ofcontacting the oligonucleotide inhibited thrombin with an antisense tothe oligonucleotide. In one embodiment, the antisense to theoligonucleotide is immobilized on a solid phase.

In some embodiments, the antisense is bound to the solid phase directlyor indirectly.

In some embodiments, the solid phase is selected from the groupconsisting of chromatographic media beads and filters.

In all aspects herein, the thrombin binding oligonucleotides are notused as anticoagulants.

In another aspect, the invention relates to a delivery applicatorcomprising:

a barrel holding an oligonucleotide inhibited thrombin; anda vessel having a delivery opening and holding an antisenseoligonucleotide linked to a solid phase;wherein the barrel and the vessel are capable of being in fluidcommunication, so that after fluid communication and contact of theinhibited thrombin with the solid phase, thrombin activity is increasedbefore delivering thrombin through the delivery opening.

In some embodiments, the fluid communication is via a re-sealableopening positioned between the barrel and the vessel.

In some embodiments, the delivery applicator further comprises a barrelhaving a delivery opening and holding fibrinogen.

In another aspect, the invention relates to a delivery applicatorcomprising:

a barrel holding an oligonucleotide inhibited thrombin; anda vessel having a delivery opening and holding an antisenseoligonucleotide;wherein the barrel and the vessel are capable of being in fluidcommunication, so that after fluid communication and contact of theinhibited thrombin with the antisense, thrombin activity is increasedbefore delivering thrombin through the delivery opening.

In another aspect, the invention relates to a delivery applicatorcomprising:

a barrel holding an oligonucleotide inhibited thrombin; and an antisenseoligonucleotide, wherein the oligonucleotide inhibited thrombin and theantisense oligonucleotide are held in separate chambers that are capableof being in fluid communication, so that allowing fluid communicationbetween the chambers and contact between the inhibited thrombin and theantisense results in increased thrombin activity before delivery, andwherein the barrel has an opening for delivery therethrough of thethrombin.

In another aspect, the invention relates to a delivery applicatorcomprising:

a container holding an oligonucleotide inhibited thrombin; and anantisense oligonucleotide linked to a solid phase, wherein theoligonucleotide inhibited thrombin and the antisense oligonucleotide areheld in separate chambers that are capable of being in fluidcommunication, so that fluid communication between the chambers andcontact between the inhibited thrombin and the solid phase results inincreased thrombin activity before delivery, and wherein the containerhas a re-sealable opening for delivery therethrough of the thrombin.

Re-sealable opening is an opening sealed by e.g. a membrane, cap,needle, rubber cap, stent, delivery tube and/or tip.

In one embodiment, a re-sealable opening prevents or minimizes leakageof liquid.

In some embodiments, the delivery applicator further comprises acontainer having a delivery opening and holding fibrinogen.

In some embodiments, the inhibited thrombin, fibrinogen and/or theformulations can be in solid, dry, aqueous, and/or frozen form. Thesolid phase can be dry or suspended in liquid, e.g. buffers.

In some embodiments the kits, device, containers, chambers, vessel,delivery application, barrel and/or syringe etc. may comprise an aqueousliquid for reconstitution.

An oligonucleotide inhibited thrombin is a thrombin combined with anoligonucleotide, said oligonucleotide can bind to thrombin in a thrombinaqueous solution, and thrombin activity is inhibited by theoligonucleotide, at least partially.

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description of theinvention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing that the antisense oligonucleotide (AS)effectively counteracts TBA1 (Thrombin Binding Aptamer 1) inhibition ofthrombin. Specifically, TBA1 inhibits 10IU/ml (˜0.1 μM) thrombin in adose dependent manner. Equimolar antisense treatment completely restoresactivity (as measured by a clotting assay).

FIG. 2A-2D are graphs representing thrombin stabilization by TBA1, overtime and at different temperatures. TBA1 was added to thrombin (˜1000IU/ml) at the indicated concentrations and incubated at RT for up to 90days (2A) at 37° C. for up to 14 days (2B) or at 2-8° C. for up to 180days (2C). The binding of TBA1 was reversed with an equimolarconcentration of antisense oligonucleotide, and diluted 100-fold fortesting the activity of the remaining thrombin tested using the clottingassay. (2D): Remaining thrombin activities after incubation at 37° C.for 7 days in the presence of different TBA1 concentrations as in FIG.2B are plotted against TBA1 concentrations (μM).

FIG. 3 is a bar graph showing similar elasticity of a control clot, anda clot formed at the presence of 25 μM TBA1+25 μM antisenseoligonucleotide.

FIGS. 4A and 4B are graphs showing that TBA1 cannot be fully reversedwith antisense within the time frame required for thrombin to clot afibrinogen solution.

FIG. 4A: TBA1 is neutralized with increasing amounts of antisenseoligonucleotides and thrombin activity tested in a thrombin activityassay. Increasing the antisense:TBA1 ratio above 1:1 results inincreasing recovery of thrombin activity, but 100% recovery cannot bereached. FIG. 4B: TBA1 is neutralized with up to 40 μM antisense(antisense:TBA1 ratios=1-4) in the same setting peaks at 60% recovery ofthrombin activity, as measured by the thrombin activity assay. In bothexperiments, thrombin activity was tested immediately after addition ofantisense oligonucleotide and mixing.

FIG. 5 is a graph showing time-dependent reversal of 25 μM TBA-mediatedinhibition with antisense oligonucleotides, with a low thrombinconcentration (˜0.1 μM). TBA inhibition at a large molar excess overthrombin (250:1) is not effectively neutralized with the antisenseoligonucleotide even after 30 minutes.

FIG. 6 is a bar graph showing validity of rapid neutralization of TBA1with antisense oligonucleotides at optimized molar ratios in a droptest. Control was performed without TBA1 or antisense oligonucleotides.Experiments were conducted using different ratios of TBA1:antisense,when the antisense was added in a separate compartments or antisense wasadded directly to the thrombin component containing TBA at twoconcentrations.

FIG. 7 is a bar graph showing that the kinetics of neutralization of 25μM aptamer by antisense oligonucleotides is effective for use in aclassical fibrin sealant setting (1000 IU/ml thrombin solution). Amodified drop test shows that a two minutes pre-incubation ofthrombin/TBA1 (25 μM) with equimolar antisense oligonucleotides issufficient to reduce inhibition to non-significant levels of thrombin(1000 IU/ml).

FIG. 8 shows that TBA1 added to the thrombin component in atwo-component fibrin sealant can be efficiently reversed with antisenseoligonucleotides added to the thrombin/TBA component in an in vivosetting. Results from the rat kidney hemostasis model show comparablehemostatic activity for thrombin (control) and TBA1-inhibited,antisense-neutralized thrombin (test). 25 μM TBA1 and antisense wereused. An equimolar amount of antisense oligonucleotide was added to thethrombin just prior to the assembly of the EVICEL® device (fitted with aspray tip extension for application). The thrombin concentration is 1000IU/ml. Full symbols: average value, open symbols: results for individualrats.

FIG. 9A shows the inhibition of thrombin mediated by TBA1 and thereversal inhibition mediated by antisense (AS) oligonucleotide bound tobiotin or to biotin-streptavidin beads. Increasing amounts of TBA1 wereadded to ˜0.1 μM (10 IU/ml) thrombin and thrombin activity was inhibitedin a dose-dependent manner (diamonds). Addition of biotinylatedantisense oligonucleotide at equimolar amounts/equal concentration toTBA1 restored thrombin activity up to ˜40% of initial activity(squares). Addition of pre-incubated biotinylated antisenseoligonucleotide with Sepharose-Streptavidin at the same concentrationsresulted in similar thrombin activity restauration like withbiotinylated TBA1 alone. Furthermore, addition of biotinylated beadsalone at the highest concentration did not inhibit thrombin activity atall (not shown).

In FIG. 9B the biotinylated antisense oligonucleotide or thebiotinylated antisense oligonucleotide pre-incubated with theSeapharose-Streptavidin was added to the reaction at least 15 minutesprior to the thrombin activity testing, thereby giving enough time forTBA1/biotinylated antisense oligonucleotide interaction, in FIG. 9B thetime needed for maximal restoration of thrombin activity was assayed:biotinylated antisense oligonucleotide pre-incubated with theSeapharose-Streptavidin was added at the indicated time points prior tothrombin activity testing. The results show that higher TBA1concentrations need longer time for reversing thrombin inhibition bybiotinylated-AS than lower concentrations of TBA1 with correspondingconcentration of biotinylated-AS.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based in part upon the finding that thrombinbinding oligonucleotides developed for treating coagulation disordersare capable of reversibly inhibiting and stabilizing thrombin activityin a liquid thrombin formulation.

The terms “reversibly stabilizing thrombin activity” and “reversiblystabilizing thrombin” refer to reducing or preventing, in part or infull, thrombin autolytic activity in a manner that can be counteractedso as to permit thrombin to carry out its biological activity onheterologous substrates, including the conversion of fibrinogen tofibrin.

The term “thrombin fragment” includes an amino acid sequence ofthrombin, linear or non-linear, that maintains thrombin activity, forexample, thrombin autolytic activity and/or thrombin mediated fibrinogencleavage.

Provided herein are methods of using isolated oligonucleotides that bindto thrombin for the inhibition and stabilization of thrombin activity.Further provided herein are thrombin formulations comprising thrombinand thrombin binding oligonucleotides and methods of reversing theinhibition of thrombin and uses thereof for converting fibrinogen tofibrin.

“Nucleotide” is meant to encompass a monomer of a nucleic acid,including but not limited to deoxyribonucleotides and/orribonucleotides, which may be natural or synthetic, and or modified orunmodified. Modifications include changes to the sugar moiety, the basemoiety and or the linkages between ribonucleotides in theoligoribonucleotide. Accordingly, as used herein, the term“deoxyribonucleotide” encompasses natural and synthetic, unmodified andmodified deoxyribonucleotides and the term “ribonucleotide” encompassesnatural and synthetic, unmodified and modified ribonucleotides.

The term “modified nucleotide” refers to nucleotides that have one ormore non-naturally occurring substituents which function in a similarmanner to natural nucleotides. Such modified nucleotides may bepreferred over the naturally occurring forms because of desirableproperties such as, for example, enhanced affinity for a target (e.g.protein target or antisense oligonucleotide) and enhanced nucleasestability. Non-limiting examples include 2′ sugar modifications such as2′O-methyl, 2′O-ethyl, 2′fluoro and the like; modified sugar moietiesincluding bridged nucleic acids (for example, LNA and ENA disclosed inPCT Patent Publication No. WO 98/39352, WO 00/47599 and WO 99/14226) andaltritol (for example, Allart, et al., 1998. Nucleosides & Nucleotides17:1523-1526; Herdewijn et al., 1999. Nucleosides & Nucleotides18:1371-1376; Fisher et al., 2007, NAR 35(4):1064-1074); modifiedinternucleotide linkages including phosphorothioate,phosphonocarboxylate and/or phosphinocarboxylate linkages (for example,U.S. Pat. Nos. 6,693,187 and 7,067,641).

Oligonucleotide” refers to a deoxyribonucleotide sequence, aribonucleotide sequence or a chimera of DNA and RNA from about 8 toabout 60 nucleotides. Each DNA or RNA nucleotide forming theoligonucleotide may be independently natural or synthetic, and may beunmodified or modified (for example as described hereinabove). Thenucleotide sequence of an oligonucleotide is written according to theconventional notation, with 5′ terminus appearing on the left hand ofthe sequence and the 3′ terminus appearing on the right hand thereof.Oligonucleotides may be provided as salts, for example a sodium salt.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by the classic Watson-Crickbonding or other non-traditional bonding. Complementarity can be full orpartial. As used herein, the thrombin binding oligonucleotide hascomplementarity to the antisense oligonucleotide.

As used herein, the term “aptamer” is a nucleic acid sequence that bindsto a molecular target such as a small molecule, a peptide, a protein,and even a microorganism such as a virus or a bacteria. Aptamers areselected by incubating the target molecule with a large pool of binderssuch as oligonucleotides (usually about 10 to 60 mers). One of themethods for selection of oligonucleotide aptamers is called “systematicevolution of ligands by exponential enrichment” (SELEX) is generallyused with modification and variations for the selection of specificaptamers.

In preferred embodiments disclosed herein, the aptamer is a DNA and/orRNA (DNA, RNA or chimera DNA/RNA) and includes unmodified and ormodified nucleotides.

In some embodiments, the aptamer or a salt of such aptamer is an RNA orDNA single-strand oligonucleotides which bind to a target protein and donot generally exhibit non-specific effects. Aptamers can be modified forstability or other desired qualities in accordance with any nucleic acidmodifications known to one of skill in the art.

Thioaptamers are aptamers which contain sulfur modifications at specificinternucleoside phosphoryl sites, and may possess enhanced stability,nuclease resistance, target affinity and/or selectivity. Examples ofthioaptamers include phosphoromonothioate (S-ODN) and phosphorodithioate(S2-ODN) oligodeoxy thioaptamers. Further information on aptamers andthioaptamers can be found in U.S. Pat. Nos. 5,218,088 and 6,423,493.

Thrombin Binding Aptamers (TBAs) are known for their potential use asanticoagulants. However, due to their short half-life in blood, theiruse has been shown not to be efficient (See Bock, et al., 1992. Nature.1992 355(6360):564-6.; Tasset and Kubik, 1997, J Mol Biol.272(5):688-98; Dougan et al., 2000 27(3):289-97; DeAnda, et al., 199458(2):344-50; Griffen et al., 1993. Blood 81:3271-76; PCT PatentPublication Nos. WO2007/025049 and WO2010/033167).

Antisense oligonucleotides are single stranded DNA or RNA molecules orchimeras of DNA and RNA or DNA or RNA analogs of about 8 to about 60nucleotides in length. As used herein, an antisense oligonucleotidecomprises a sequence that is complementary to the thrombin bindingoligonucleotide. The antisense oligonucleotide can be of the same lengthas the thrombin binding oligonucleotide. In some embodiments, theantisense oligonucleotides comprise additional residues, or may be afragment thereof as long as they retain the binding affinity to thethrombin binding oligonucleotide to reverse the binding of the thrombinbinding oligonucleotide to thrombin. The antisense oligonucleotide canfurther be covalently or non-covalently attached to, or associated with,a nucleotide or non-nucleotide moiety or a molecule which may includeone or more of, e.g. a nucleotide, a nucleotide analog, an amino acid, apeptide, a polypeptide, a lipid moiety, a carbohydrate moiety, a marker,a matrix, any kind of beads or a tag. The antisense can be bound to theabove molecules through a linker such as, but not limited to variouslengths poly-A or poly-T oligonucleotide chains, polypeptide chains,aliphatic chains. In one embodiment, the linker will decrease stericinterference.

The antisense oligonucleotide may further include one or moremodifications, as described above.

In some embodiments, the antisense oligonucleotide sequence comprisesany one of SEQ ID NO:7-12. In some embodiments, the antisenseoligonucleotide sequence is set forth in any one of SEQ ID NO:7-12. Insome embodiments, the antisense oligonucleotide is a salt, for example asodium salt. In some embodiments, the antisense oligonucleotide isprovided as a solution comprising a pharmaceutically acceptable carrier.

In one aspect, the invention relates to a method for reversing anoligonucleotide inhibited thrombin, the method comprising the steps ofcontacting the oligonucleotide inhibited thrombin with an antisense tothe oligonucleotide, wherein the antisense to the oligonucleotide isimmobilized on a solid phase.

Subject of the present invention is also a solid phase or supportcovalently bound to an antisense oligonucleotide of a thrombin-bindingoligonucleotide.

The support is preferably a chromatographic material which is able tobind the antisense oligonucleotide. The chromatographic material to beemployed according to the method of the invention is e.g. a hydrophilicmaterial such as agarose, cellulose, controlled pore glass, silica gels,dextrans or ceramic material or an organic artificial polymer such asbased on polyacrylamides polystyrens. Typical materials are commerciallyavailable under the trade names Agarose, Sephadex®, Sepharose®,Sephacryl® (GE, Sweden), Ultragel® (Biosepara, France), TSK-Gel,Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deerfield, Ill.,USA), Eupergit® (Rohm Pharma, Darmstadt, Germany), Fractogel®, Eshmuno®(Merck Millipore, USA), Ceramic HyperD® (Pall, USA). Also materialsbased on azlactones (3M, St. Paul, Minn., USA) can be used.

An embodiment employs a particulate chromatographic material or amonolithic block-material. The particulate material can be suspended inan appropriate medium and the resulting slurry can be used e.g. in achromatographic column. However, the material can be used in a batch.

In one embodiment, the antisense is bound to a support preferably via alinker, in particular a bifunctional linker, between the support and theantisense. If a bifunctional linker is used, it can be selected from thegroup consisting of N-hydroxy succinimide, DAPA, CNBr, epoxy,diaminodipropylamine (DADPA), 1,6 diaminohexane, succinic acid, 1,3diamino-2-propanol, ethylendiamine (EDA), TNB, pyridyldisulfide,iodoacetamide, maleimide activated support or combinations thereof.

The support for performing the method of the invention is can bemodified by a moiety which reacts with primary or secondary aminogroups.

Further, a support or solid phase having the antisense covalently boundis also subject of the present invention. The support of the inventionis preferably a chromatographic material, such as a hydrophilicchromatographic material such as dextran or an organic artificialpolymer such as mentioned above.

The chromatographic material which forms the support may be aparticulate material or a monolithic block-material. E.g, the latter isdescribed in Hermanson et al, incorporated by reference (Hermanson GT,Mallia AK and Smith PK 1992 “Immobilization Affinity Ligand Techniques”pp. 454 Academic Press, Inc. San Diego, USA).

In another preferred embodiment of the invention the antisense is boundto the support via a linker between the support and the antisense. Thisis advantageous when the support does not have functional groups beingcapable to bind the antisense covalently. Then the support is firstfunctionalized and then reacted with a linker which is able to bind theantisense. Spacer arms or leashes are low molecular weight moleculesthat are used as intermediary linkers between a support or matrix andaffinity ligand such as the antisense. Preferably, the spacers comprisetwo functional groups on both ends for easy coupling to ligand andsupport. The spacer is typically a hydrocarbon compound having twofunctional groups at its ends. One of the two ends is attachedcovalently to the matrix using conventional or per se known reactions.The second end is covalently linked to the ligand using another couplingprocedure.

Example of are a bifunctional linker such as N-hydroxy succinimide,DAPA, CNBr, epoxy, diaminodipropylamine (DADPA), 1,6 diaminohexane,succinic acid, 1,3 diamino-2-propanol, ethylendiamine (EDA), TNB,pyridyldisulfide, iodoacetamide, maleimide activated support orcombinations thereof.

Since many functionalized supports are commercially available, it may beadvantageous to start with a support which is modified by a moiety whichreacts with primary or secondary amino groups.

An oligonucleotide inhibited thrombin is a thrombin combined with anoligonucleotide, said oligonucleotide can bind to thrombin in a thrombinaqueous solution, and thrombin activity is inhibited by theoligonucleotide, at least partially.

Immobilized antisense oligonucleotides to a solid phase or support areantisense oligonucleotides that are bound covalently and/ornon-covalently to a solid phase or support. Examples of covalent bindingantisense to a solid phase were described above. In one embodiment, theantisense is bound to the solid phase via non covalent binding. In oneExample, a solid phase is covalently bound to streptavidin and theantisense oligonucleotide is covalently bound to biotin. Thebiotinylated antisense is non-covalently bound to streptavidin linkedsolid phase via biotin streptavidin affinity binding.

Different amounts (i.e. concentrations) of antisense can be bound on asupport. Increase in antisense concentration on the support mayaccelerate reversal of inhibition resulting in an increase of thrombinactivity/reactivation.

Typically, an increase of thrombin activity following inhibition withthe oligonucleotide by using an antisense oligonucleotide (e.g. aptamer)is referred to re-activation of thrombin.

Re-activated thrombin , is thrombin having increased activity comparedto the activity of oligonucleotide inhibited thrombin.

Re-activated thrombin can also refer to thrombin having increasedactivity compared to activity of thrombin after it is combined with theoligonucleotide capable of inhibiting and binding thrombin.

Using an antisense immobilized on a support (e.g. filter) with a device,container, chamber, vessel, delivery applicator, barrel, syringe etc.allows to deliver the re-activated thrombin.

The device may also include a mesh to prevent or minimize passage of thesupport through an opening, such as a delivery opening.

In some embodiments the device comprises at least one mesh; the mesh isconfigured to retain the beads within the device.

In some embodiments, the mesh size (i.e. the size of the pores therein)is at least 1.5-fold smaller (e.g. 2-fold smaller) than the size of thesmallest bead. Non limiting examples of meshes are grids, etchedmaterials, polymer networks, and the like. Meshes can be composed of anymaterial e.g. biocompatible material such as plastic, nylon, cellulose,alloys, glass and the like. The device can comprise more than one meshelement. Filter paper is one example of a mesh—in different embodiments;other mesh structures (i.e. other than filter paper) can be used.

An oligonucleotide (e.g. aptamer) inhibited thrombin held in a container(e.g. a syringe) could be mixed with the antisense oligonucleotide (e.g.antisense of aptamer), the antisense can be bound on a solid phase orsupport (e.g. beads), to re-activate thrombin, at least partially, andthe thrombin after re-activation can be applied (e.g. expelled from theopening of a syringe) on a desired surface. In case of using antisenseoligonucleotide (e.g. antisense of aptamer) bound on a solid phase orsupport (e.g. beads), before application (e.g. before expelling from theopening of a syringe) the beads are separated from the mixture e.g. byfiltration, precipitation over time by the gravitation force and/orfollowing centrifugation.

For example separation/removal can be achieved by using at least onemesh e.g. positioned on the delivery opening and/or before the deliveryopening.

Alternatively or in addition, the beads can be separated/removed from aliquid or from a mixture by decantation.

Typically, decantation is a process for the separation of mixtures byremoving a layer of liquid, generally from which a precipitate hassettled.

A precipitate can be beads bound to the antisense oligonucleotide. Theantisense bound to the beads can be complexed with the oligonucleotidecapable of binding thrombin.

The beads can be sedimented e.g. centrifuged by 1 min spin down atapproximately 5000 g and supernatant containing the re-activatedthrombin can be collected.

A “surface” is a position or location where one desires to apply there-activated thrombin e.g. a bleeding tissue or wound on subject inneed. The surface depends on the use of the re-activated thrombin. There-activated thrombin may be used e.g. in combination with fibrinogen,for example, in hemostasis, tissue fixation, graft fixation, woundhealing and anastomosis.

Provided is a delivery applicator comprising:

a barrel holding an oligonucleotide inhibited thrombin; anda vessel having a delivery opening and holding an antisenseoligonucleotide linked to a solid phase;wherein the barrel and the vessel are capable of being in fluidcommunication, so that after fluid communication and contact of theinhibited thrombin with the solid phase, thrombin activity isincreased/re-activated , before delivering thrombin through the deliveryopening.

The barrel and vessel of the device can be in fluid communication via aresealable opening between the barrel and vessel. Provided, is adelivery applicator comprising: a container holding an oligonucleotideinhibited thrombin; and an antisense oligonucleotide linked to a solidphase, wherein the oligonucleotide inhibited thrombin and the antisenseoligonucleotide are held in separate chambers that are capable of beingin fluid communication, so that fluid communication between the chambersand contact between the inhibited thrombin and the solid phase resultsin increased thrombin, or reactivated thrombin, activity beforedelivery, and wherein the barrel has a re-sealable opening for deliverytherethrough of the thrombin.

Increased thrombin activity is in comparison to the activity of thethrombin while being inhibited by the oligonucleotide.

In one embodiment, the chambers are divided by a septum, which is atleast partially breakable, braking the septum allows mixing theoligonucleotide inhibited thrombin and the antisense oligonucleotidebefore administration.

In some embodiments, the container and/or chambers are flexible andbreaking the septum can be achieved by applying pressure onto thecontainer and/or chambers.

The size of each chamber and fill volumes are dependent e.g. on theintended use, suitable concentration ratios between the antisenseoligonucleotides and the thrombin binding oligonucelotide, and/ordesired volume.

The delivery opening of the container and/or chamber can be a Male orFemale Luer Lock.

In some embodiments, the container comprises two or more chambers asdescribed in WO1997042897A1 e.g. FIG. 19.

“Breakable” can be interchangeable with the term “peelable” and“frangible”.

The term “contacting” refers to a combining action which e.g. brings theoligonucleotide inhibited thrombin into contact with the antisense in amanner that a binding interaction will occur between the antisense andthe oligonucleotide and/or brings the oligonucleotide into contact withthrombin in a manner that allows a binding interaction between thethrombin and the oligonucleotide.

The term “contacting” includes the term “adding” and the term“addition”.

The combining action can be for a sufficient period of time which allowscontacting, binding and/or complexing e.g. between the antisense and theoligonucleotide and/or the thrombin and the oligonucleotide.

In some embodiments, the antisense is bound to the solid phase directlyor indirectly.

In some embodiments, the solid phase is selected from the groupconsisting of chromatographic media beads and filters. Chromatographicmedia (beads) may include, but are not limited to cross linked agarose,cross linked dextran, methacrylic, polyvinyl, silica based materials.These may be charged and/or modified to allow covalent or non-covalentbinding of antisense nucleotides. Bead grade (size) can range fromsuperfine (20 micron mean size) and up to coarse (150 micron mean size).

Filters may include, but are not limited to, PVDF, polypropylene, nylonbased. In some embodiments, the filters may have a membrane or depthconstruction. These may be charged and/or modified to allow covalent ornon-covalent binding of antisense nucleotides. Filter porosity (rating)can range from 0.45 micron and up to about 20 micron.

“Thrombin” or “thrombin polypeptide” is a mammalian serine proteasewhich is part of the blood coagulation cascade and converts fibrinogeninto insoluble strands of fibrin, as well as catalyzes othercoagulation-related reactions. In humans, prothrombin is encoded by theF2 gene, and the resulting polypeptide is proteolytically cleaved in thecoagulation cascade to form thrombin. Thrombin serves, inter alia, as anactive component in several hemostasis products. For example, fibrinsealants typically comprise a fibrinogen component and a thrombincomponent. When both components are mixed (e.g. when applied to ableeding wound) thrombin cleaves fibrinogen and a fibrin polymer isformed.

Thrombin is a serine protease which results from the cleavage ofprothrombin (Factor II), a zymogen precursor, by another serine protease(Factor Xa). Human thrombin is a 295 amino acid protein composed of twopolypeptide chains joined by a disulfide bond.

The zymogen is cleaved at residue 155 and residue 271, removing theentire N-terminal 271 amino acids. An additional intramolecular cleavageby Factor Xa at residue 320 yields the active alpha thrombin moleculewhich is a 295 amino acid polypeptide (human) composed of a heavy andlight chain held together via a single S—S bond (Krishnaswamy J, (2013)“The transition of prothrombin to thrombin”. J Thromb Haemost. June; 11Suppl 1:265-76). Thrombin, being a serine protease, can initiate its owndegradation (“autolysis”) by cleaving other thrombin molecules at thebeta (residue 382 and 394) or gamma (residue 443 and residue 474) sites,yielding beta- and gamma-thrombin, respectively. Neither of these loopscontains a classic thrombin recognition site, nor is this cleavagespecific to a certain residue within the loops. Rather, these loops areboth flexible and exposed and are cleaved for lack of a proper substrateand especially at high thrombin concentration (see for example, Chang, JY. Biochem. J. (1986) 240:797-802, “The structures and proteolyticspecificities of autolysed human thrombin”; Rydel T J, et al., J BiolChem. 1994, 269(35):22000-6. Crystallographic structure of humangamma-thrombin”; Pozzi N, et al., Biophys Chem. 2011, 159(1):6-13“Rigidification of the autolysis loop enhances Na(+) binding tothrombin”). The inactivation of thrombin in-vivo does not proceed viathis mechanism (autolysis) but rather via a specific interaction(bridged by heparin) with the serine protease inhibitor (SERPIN),anti-thrombin III (ATIII). The interaction of thrombin (and severalother homologous serine proteases such as Factor X and even protein C)with ATIII is mediated via the gamma loop (see, for example, Yang, L.,Blood. 2004, 104(6):1753-9, “Heparin-activated antithrombin interactswith the autolysis loop of target coagulation proteases”; and Marino, F,J Biol Chem. 2010, 285(25):19145-52. “Engineering thrombin for selectivespecificity toward protein C and PAR1”).

Human, non-human, recombinant or non-recombinant thrombin can be usedwithin the present invention. Thrombin is used medically e.g. as ahemostatic agent and as a component of tissue adhesives.

“Thrombin activity” is meant to include thrombin mediated conversion ofheterologous substrates, including proteins e.g. fibrinogen into fibrin,as well as the conversion of Factor VIII to Factor VIIIa, XI to XIa,XIII to XIIIa, and Factor V to Va.

A “heterologous substrate” is a substrate, preferably a proteinsubstrate, other than thrombin. In some embodiments, the thrombinactivity refers to conversion of fibrinogen into fibrin.

The term “stabilizing” means, for example, maintaining the thrombinactivity within the thrombin liquid solution at a level of about 70% toabout 100% (e.g. about 80% to 100% or 90% to 100%) compared to theinitial thrombin activity e.g. after one, 2, 3, 6, 9 and up to 12 monthsat room temperature and/or 2, 3, 4 weeks and up to 1 month at 37° C.and/or 3, 6, 9, 12, 18 and up to 24 months at 2-8° C. in liquid form. Athrombin solution is stable when, for example, autolytic and otherprotease activity is minimal or absent. The term “inhibiting thrombinactivity” means, for example, preventing, partially or fully, thrombinautolysis and/or cleavage of a thrombin substrate, for examplefibrinogen. In some embodiments, inhibiting thrombin activity refers topreventing or reducing thrombin autolysis in an aqueous liquid thrombinsolution, so that about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or preferablygreater than 90% uncleaved thrombin remains in the solution.

For long-term storage, the formulation, comprising the thrombin and thethrombin binding oligonucleotide, is aliquoted into sterile vials,ampoules, or other containers, which are then sealed. In one embodiment,a seal that permits removal of the stabilized thrombin composition witha syringe through the seal is used. The container is labeled accordingto standard practice in the pharmaceutical or medical device field.

In some embodiments, the container is provided in a kit with a secondcontainer containing an antisense oligonucleotide. In anotherembodiment, the container is provided in a kit with yet a thirdcontainer comprising a fibrinogen comprising component. The kit mayfurther comprise an application device, such as a sprayer, syringe, orthe like and/or a diluent and/or instructions for use.

For use, the stabilized thrombin formulation, e.g. an aqueous liquidthrombin formulation comprising a thrombin and a thrombin bindingoligonucleotide, can be used directly from the container or can befurther diluted to the desired concentration, generally the thrombinactivity in the formulation is from about 1 IU/ml to about 15,000 IU/ml,about 20 IU/ml to 15,000 IU/ml, or 100 IU/ml to 5,000 IU/ml, 200 IU/mlto about 1000 IU/ml or about 300 IU/ml to about 1000 IU/ml, although theactual concentration will be determined by the user (e.g. medicalattendant, physician, nurse, medic) i.e. according to the needs of theindividual patient and on the severity of bleeding. The stabilizedthrombin formulation can be applied to bleeding tissue to achievehemostasis, per se or may be used in combination with a scaffold ormatrix, for example an absorbable scaffold or matrix. The stabilizedthrombin formulation can also be used as a component of tissue adhesive,fibrin sealant or fibrin glue. These and other known in the art uses ofthrombin formulation are contemplated for the disclosed stabilizedthrombin. Numerous uses of fibrin glue in various fields have beenreported, including use as a sealant e.g. for sealing leaks, hemostaticagent/stop bleeding, adhesion prevention, to enhance healing, forjoining structures, in a variety of open and laparoscopic surgeries.

Preferred hemostatic scaffolds are natural or genetically engineeredabsorbable polymers or synthetic absorbable polymers, or mixturesthereof. Examples of natural or genetically engineered absorbablepolymers are proteins, polysaccharides and combinations thereof.Proteins include, prothrombin, thrombin, fibrinogen, fibrin,fibronectin, heparinase, Factor X/Xa, Factor VIINIIa, Factor IX/IXa,Factor XI/XIa, Factor XII/XIIa, tissue factor, batroxobin, ancrod,ecarin, von Willebrand Factor, collagen, elastin, albumin, gelatin,platelet surface glycoproteins, vasopressin, vasopressin analogs,epinephrine, selectin, procoagulant venom, plasminogen activatorinhibitor, platelet activating agents, synthetic peptides havinghemostatic activity, and/or combinations thereof. Polysaccharidesinclude, without limitation, cellulose, alkyl cellulose, e.g.methylcellulose, alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose,cellulose sulfate, salts of carboxymethyl cellulose, carboxymethylcellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin,hyaluronic acid, salts of hyaluronic acid, alginate, alginic acid,propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan,pectin, pullulan, xanthan, chondroitin, chondroitin sulfates,carboxymethyl dextran, carboxymethyl chitosan, chitosan, heparin,heparin sulfate, heparan, heparan sulfate, dermatan sulfate, keratansulfate, carrageenans, chitosan, starch, amylose, amylopectin,polyN-glucosamine, polymannuronic acid, polyglucuronic acid, andderivatives of any of the above. Examples of synthetic absorbablepolymers are aliphatic polyester polymers, copolymers, and/orcombinations thereof.

As used herein, the indefinite articles “a” and “an” mean “at least one”or “one or more” unless the context clearly dictates otherwise.

As used herein, the terms “comprising”, “including”, “having” andgrammatical variants thereof are to be taken as specifying the statedfeatures, steps or components but do not preclude the addition of one ormore additional features, steps, components or groups thereof.

When a numerical value is preceded by the term “about”, the term “about”is intended to indicate +/−10%.

A “polynucleotide coding sequence” or a sequence that “encodes” aselected polypeptide, is a nucleic acid molecule that is transcribedinto DNA or RNA or transcribed and translated into a polypeptide in vivowhen placed under the control of appropriate regulatory sequences (or“control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxyl) terminus. A transcription terminationsequence may be located 3′ to the coding sequence. Typical “controlelements”, include, but are not limited to, transcription regulators,such as promoters, transcription enhancer elements, transcriptiontermination signals, and polyadenylation sequences; and translationregulators, such as sequences for optimization of initiation oftranslation, e.g., Shine-Dalgarno (ribosome binding site) sequences,Kozak sequences (i.e., sequences for the optimization of translation,located, for example, 5′ to the coding sequence), leader sequences(heterologous or native), translation initiation codon (e.g., ATG), andtranslation termination sequences. Promoters can include induciblepromoters (where expression of a polynucleotide sequence operably linkedto the promoter is induced by an analyte, cofactor, regulatory protein,etc.), repressible promoters (where expression of a polynucleotidesequence operably linked to the promoter is included by an analyte,cofactor, regulatory protein, etc.), and constitutive promoters.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

A “recombinant” nucleic acid molecule as used herein to describe anucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. “Recombinant host cells”, “host cells”, “cells”, “celllines”, “cell cultures”, and other such terms denoting prokaryoticmicroorganisms or eukaryotic cell lines cultured as unicellularentities, are used interchangeably, and refer to cells which can be, orhave been, used as recipients for constructs, vectors or other transferDNA, and include the progeny of the original cell which has beentransfected. It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement to the original parent, due to accidental ordeliberate mutation. Progeny of the parental cell which are sufficientlysimilar to the parent to be characterized by the relevant property, suchas the presence of a nucleotide sequence encoding a desired peptide, areincluded in the progeny intended by this definition, and are covered bythe above terms.

The oligonucleotides disclosed herein may be chemically synthesized orrecombinantly produced. DNA and RNA oligonucleotides, including chimerasof RNA and DNA or RNA and/or DNA analogs, may be synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19; PCT Publication No. WO99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684,Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al., 1998,Biotechnol Bioeng., 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311;Usman et al., 1987, J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990,Nucleic Acids Res., 18, 5433; all incorporated herein by reference.

In one embodiment, the oligonucleotides disclosed herein are chemicallysynthesized. In other embodiments, the oligonucleotides disclosed hereinare produced in-vivo or ex-vivo by expression of recombinant DNA inprokaryotic or eukaryotic host cells to generate DNA and/or RNAoligonucleotides. In various embodiments, provided is a recombinantpeptide encoded by an isolated nucleic acid sequence. In someembodiments, the thrombin binding oligonucleotide comprises a nucleicacid sequence set forth in any one of SEQ ID NOS: 1 to 6. In someembodiments, the antisense oligonucleotide comprises a nucleic acidsequence set forth in any one of SEQ ID NOS: 7 to 12. Accordingly,provided herein is a vector comprising the nucleic acid sequence used togenerate a DNA or RNA oligonucleotide, operatively linked to a promoterelement. Further provided is a host cell comprising such vector.

As used herein, an oligonucleotide or aptamer is said to “interact” withor “bind” to a protein (e.g. thrombin binding oligonucleotide withthrombin) if it associates with protein preferably via non-covalentbinding forces, for example van der Waals and electrostatic forces.

“Room temperature” is meant to include temperature of about 20° C. toabout 28° C., or 22° C. to about 26° C.

As used herein the terms “autolysis” or “auto degradation” refer to theunfavorable molecular degradation of thrombin into an inactive orpartially active form.

A preferred thrombin binding oligonucleotide as disclosed herein, iscapable of reversibly inhibiting thrombin activity, for example, byreducing thrombin autolysis and thrombin activity towards fibrinogenwherein the inhibition is reversible with an antisense oligonucleotide.Without wishing to be bound to theory, the antisense oligonucleotide hasstronger binding affinity to the thrombin binding oligonucleotide thanthrombin to the thrombin binding oligonucleotide.

The term “affinity” refers to the strength of binding and can beexpressed quantitatively as a dissociation constant (Kd).

In one embodiment, the thrombin binding oligonucleotide interacts withthe antisense oligonucleotide disclosed herein with at least 1.10, 1,15,1.20, 1.25, 1.30, 1.35, 1.40, 1.45, 1.50, 1.55, 2 fold greater affinity,more preferably at least 5 fold greater affinity and even morepreferably at least 10, fold greater affinity than it interacts withthrombin. Binding affinity (i.e., Kd) can be determined using standardtechniques.

The term “an effective amount” refers to the amount of an antisenseoligonucleotide disclosed herein required to bind the thrombin bindingoligonucleotide and reverse the inhibition of thrombin activity.

The “pharmaceutically acceptable” or “pharmacologically acceptable”carriers, solvents, diluents, excipients, and vehicles generally referto inert, non-toxic solid or liquid fillers, diluents or encapsulatingmaterial not reacting with the active ingredients of the compositionsdisclosed herein. Acceptable excipients include, without limitation,saline; acetic acid or acetate; calcium, sodium and chloride ions;mannitol; albumin; or combination thereof.

The invention provides oligonucleotides useful in practicing the presentinvention.

Provided herein are compounds and methods for stabilization of thrombinactivity in liquid thrombin formulations, wherein stabilizing thethrombin activity refers, for example, to reducing or preventingautolytic and biological activity. Details of the exemplaryoligonucleotides useful in practicing the invention, are provided inExample 1, hereinbelow and in the sequence listing, incorporatedherewith.

Provided herein are methods of screening for oligonucleotides capable ofreversibly stabilizing thrombin activity. Accordingly, provided is amethod for screening for an oligonucleotide capable of inhibiting andthereby stabilizing the activity of thrombin in a liquid thrombinformulation, comprising

-   -   a. contacting thrombin or a fragment thereof, each exhibiting an        initial activity of 4 to 15,000 IU/ml, with a set of test        thrombin binding oligonucleotides and identifying one or more        thrombin binding oligonucleotides which inhibit, at least        partially, the initial activity; and    -   b. contacting the thrombin bound oligonucleotide of step a) with        a set of test antisense oligonucleotides;        whereby restoration of at least 4 IU/ml thrombin activity by an        oligonucleotide antisense following step (b) indicates: 1) a        potential thrombin binding oligonucleotide for thrombin        stabilization; and 2) a potential antisense oligonucleotide to        reverse the inhibitory effect of the thrombin binding        oligonucleotide.

In some embodiments of the screening method, the thrombin has an initialactivity of 4 to 15,000 IU/ml, about 20 IU/ml to 15,000 IU/ml, or 100IU/ml to 5,000 IU/ml, 200 IU/ml to about 1000 IU/ml or about 300 IU/mlto about 1000 IU/ml.

In some embodiments the antisense oligonucleotide restores thrombinactivity to at least 4 IU/ml, at least about 20 IU/ml, at least about100 IU/ml or at least about 300 IU/ml, at least about 1000 IU/ml and upto 1500 IU/ml of the initial activity of thrombin.

Alternatively, provided is a method for screening for an oligonucleotidecapable of inhibiting and thereby stabilizing the activity of thrombinin an aqueous liquid thrombin formulation, comprising

-   -   a. contacting thrombin or a fragment thereof, each exhibiting an        initial activity, with a set of test thrombin binding        oligonucleotides and identifying one or more thrombin binding        oligonucleotides which inhibit at least 60% of the initial        activity; and    -   b. contacting a thrombin bound oligonucleotide of step a) with a        set of test antisense oligonucleotides;        whereby restoration of more than 40% of the initial activity by        an oligonucleotide antisense following step (b) indicates: 1) a        potential thrombin binding oligonucleotide for thrombin        stabilization; and 2) a potential antisense oligonucleotide to        reverse the inhibitory effect of the thrombin binding        oligonucleotide.

In some embodiments of the method, the one or more thrombin bindingoligonucleotides inhibit, at least 60% of the initial activity ofthrombin, at least 70%, at least 75%, at least 80%, at least 85% or atleast 90% of the initial thrombin activity.

In some embodiments of the method the antisense oligonucleotide restoresat least, 40%, 50% , 60%, 70% and up to 100% of the initial activity ofthrombin.

In some embodiments, the methods further include the step of isolatingthe one or more test thrombin binding oligonucleotides and/or of testingthe one or more test antisense oligonucleotides its ability to inhibitand stabilize thrombin activity.

In some embodiments of the methods, the set of test thrombin bindingoligonucleotides includes one or more test thrombin bindingoligonucleotides and the set of test antisense oligonucleotides includesone or more test antisense oligonucleotides.

The fibrinogen can be prepared from initial blood composition. The bloodcomposition can be whole blood or blood fractions, i.e. a product ofwhole blood such as plasma. Fibrinogen can be autologous, humanincluding pooled plasma, or of non-human source. It is also possiblethat the fibrinogen is prepared by recombinant methods or can bechemically modified.

In one embodiment of the invention, the fibrinogen solution is comprisedfrom a biologically active component (BAC) which is a solution ofproteins derived from blood plasma which can further comprise antifibrinolytic agents such as tranexamic acid and/or stabilizers such asarginine, lysine, their pharmaceutically acceptable salts, or mixturesthereof. BAC can be derived from cryoprecipitate, in particularconcentrated cryoprecipitate.

The term “cryoprecipitate” refers to a blood component which is obtainedfrom frozen plasma prepared from whole blood. A cryoprecipitate can beobtained when frozen plasma is thawed in the cold, typically at atemperature of 0-4° C., resulting in the formation of precipitate thatcontains fibrinogen and factor XIII. The precipitate can be collected,for example by centrifugation and dissolved in a suitable buffer such asa buffer containing 120 mM sodium chloride, 10 mM trisodium citrate, 120mM glycine, 95 mM arginine hydrochloride. The solution of BAC cancomprise additional factors such as for example factor VIII,fibronectin, von Willebrand factor (vWF), vitronectin, etc. for exampleas described in U.S. Pat. No. 6,121,232 and WO9833533. The compositionof BAC can comprise stabilizers such as tranexamic acid and argininehydrochloride. The amount of tranexamic acid in the solution of BAC canbe from about 80 to about 110 mg/ml.

In another embodiment, the concentration of plasminogen and plasmin inthe BAC composition is lowered to equal or less than 15 μg/ml like forexample 5 μg/ml or less plasminogen e.g. using a method as described inU.S. Pat. No. 7,125,569, EP 1,390,485 and WO02095019. In anotherembodiment of the invention, when the concentration of plasminogen andplasmin in the BAC composition is lowered, the composition does notcontain tranexamic acid or aprotinin.

The fibrinogen solution may be the BAC2 component (from EVICEL®) or anyother fibrinogen containing solution, such as purified recombinantfibrinogen or cryoprecipitate produced from human plasma.

While the following examples demonstrate certain embodiments of theinvention, they are not to be interpreted as limiting the scope of theinvention, but rather as contributing to a complete description of theinvention.

EXAMPLES Example 1: Thrombin Binding Oligonucleotides

Exemplary thrombin binding oligonucleotides were identified in Bock etal., Nature. 1992. 355(6360):564-6 and Tasset and Kubik,1997, J MolBiol. 272(5):688-98. For simplicity, these oligonucleotides weredesignated herein as Thrombin Binding Aptamer 1 and 2 (TBA1 (Bock,1992), TBA2 (Tasset, 1997)), respectively. TBA1, BOCK-15, which binds toEXOSITE 1 of thrombin has a nucleic acid sequence set forth in SEQ IDNO:1 5′-GGTTGGTGTGGTTGG or an extension of SEQ ID NO:1 set forth in SEQID NO:2 5′-GGGTTGGGTGTGGGTTGGG. TBA2, TASSET-29, which binds to EXOSITEII thrombin has a nucleic acid sequence set forth in SEQ ID NO:3 5′-AGTCCGTGGTAGGGCAGGTTGGGGTGACT. RNA counterparts of the thrombin bindingoligonucleotides are set forth in SEQ ID NOS:4-6.

The thrombin binding aptamers have a short half-life in the blood(Dougan, et al., Nucl Med Biol. 2000. 27(3):289-97) and at least TBA1has been shown to be safe (DeAnda et al., Ann Thorac Surg. 1994.58(2):344-50).

Exemplary thrombin binding oligonucleotides and antisenseoligonucleotides disclosed herein are provided in the appended sequencelisting

Example 2: Stabilization of Thrombin Activity

Aqueous liquid thrombin, in its purified and concentrated form 1000international units [IU]/ml and about 0.3 mg/ml thrombin, rapidlyundergoes autolysis at room temperature (RT) causing a significant lossof activity. Therefore, aqueous liquid thrombin activity is reduced whenincubated at room temperature for prolonged periods of time (e.g. after72 to 144 hours) inter alia, due to autolytic degradation. The decreaseof thrombin stability in aqueous liquid solution can be assessed bymeasuring thrombin activity after prolonged periods of time underpermissive temperature (e.g. RT or 37° C.).

Thrombin activity—clotting assay: Briefly, a thrombin standard curve wascreated using between 4-10 International Units (IU)/ml using an in housevalidated standard solution. Higher concentrations of thrombin resultedin unmeasurably fast fibrin clotting times and therefore, respectivethrombin concentrations were extrapolated from the standard curve.

Clotting time was assessed by adding 40 μl thrombin to 160 μl of a 0.1%purified commercial fibrinogen solution in a test cuvette. The time toclot was assayed in an automated clotting machine (Stat4, StagoDiagnostica). The machine generates an oscillating electromagnetic fieldwhich moves a small metal ball inside the cuvette. Clotting wasdetermined to have occurred when the ball movement stopped. Thrombinconcentration in test samples was extrapolated from the clotting timesagainst the standard curve.

The physical properties of fibrin clots were tested by measuring Young'sModulus (in kPa) using a Lloyd Instruments LFplus machine. Briefly, 8IU/ml thrombin were incubated in a cast mold with an equivalent volumeof a 7% fibrinogen containing solution (BAC2), yielding 4 IU/ml and 3.5%concentrations of thrombin and fibrinogen, respectively, for 30 minutesat 37° C. After 30 minutes, the clot was placed in the machine andsubjected to pulling at increasing force. The slope of the stretching ofthe clot as a function of the pulling force (Young's Modulus) wasextrapolated.

Thrombin activity to form fibrin sealant—drop test assay: In this assay,one barrel of a two-component fibrin sealant device, was filled with aspecified amount of thrombin (typically 200 IU/ml thrombin [˜2 μM]) andthe second barrel was filled with standard BAC2-fibrinogen solution (˜7%fibrinogen). The device was placed above a tilted plane (˜15-30°) onwhich millimeter paper was placed. A mechanized lever pushed bothbarrels simultaneously, expressing equal and measured amounts of bothsolutions through a conjoined tip. The drop containing the mix of thetwo components trickled down the slope until a clot formed. The distancetraveled by the drop was measured, being a function of the rate offibrinogen polymerization and, thereby of the effective concentration ofthrombin used.

Reversible inhibition of thrombin activity by TBA. TBA1 was shown toreversibly inhibit thrombin activity in a dose-dependent manner. A stocksolution of 10 IU/ml thrombin which is approximately equivalent to 0.1μM was prepared. TBA1 at increasing concentrations ranging fromequimolar amount (0.1 μM) and up to 10-fold higher (1 μM) was added tothe thrombin, resulting in dose-dependent inhibition of thrombinactivity (FIG. 1, dotted line). The concept of reversible inhibition ofthrombin relies on the possibility of an antisense oligonucleotide toeffectively counteract TBA1 inhibition of thrombin. Antisenseoligonucleotides (AS) SEQ ID No: 7 to TBA1 SEQ ID NO: 1 -were added atequimolar amounts to the TBA1, and allowed to incubate for 5-30 minutes.This solution was used in the thrombin activity assay in order to assessthe efficiency of the antisense neutralization of TBA1. Indeed, theneutralization was efficient as is evident by the fact that theextrapolated activity from this assay remained 10 IU/ml (solid line inFIG. 1). In FIG. 1, the x-axis is concentration of TBA1 and antisense inmicromoles. Taken together, antisense oligonucleotides effectivelycounter the TBA1 induced inhibition of thrombin and TBA1 inhibits 10IU/ml (˜0.1 μM) thrombin in a dose dependent manner. Equimolar antisensecompletely restores activity.

Example 3: Activity of Thrombin with Thrombin Binding Oligonucleotideand Antisense Oligonucleotide

Samples of 1000 IU/ml purified thrombin were stabilized with increasingfinal concentrations of TBA1 (SEQ ID NO: 1) from 1 μM and up to 40 μM(using a volume ratio of 1:10 TBA1:thrombin). The final thrombinconcentration in the mixed samples was 9 μM. The samples were incubatedfor up to 90 days at room temperature (RT) [FIG. 2A) or 180 days at 2-8°C. [FIG. 2C).

Samples of 1000 IU/ml purified thrombin were stabilized with increasingfinal concentrations of TBA1 (SEQ ID NO: 1) from 1 μM and up to 25 μM(using a volume ratio of 1:10 TBA1:thrombin). The final thrombinconcentration in the mixed samples was 9 μM. The samples were incubatedfor up 14 days at 37° C. [FIG. 2B].

In order to assess the activity of the inhibited thrombin, thethrombin/TBA1 mix was incubated with an antisense oligonucleotide (SEQID NO:7) at equivalent concentrations to TBA1 for 30 minutes. Thesamples were then diluted 100-fold for testing within the range of thethrombin clotting assay (4-10 IU/ml) in the test buffer, and assayed foractivity as before.

FIGS. 2A-2D show that TBA1 clearly stabilized thrombin in adose-dependent manner. FIGS. 2A, 2B and 2C show percent thrombinremaining when TBA1 was added to purified thrombin (as in the thrombincomponent of EVICEL® Fibrin sealant, ˜1000 IU/ml) at the indicatedconcentrations and incubated at RT for up to 90 days (FIG. 2A) at 37° C.for up to 14 days (FIG. 2B) and at 2-8° C. for 180 days (FIG. 2C).

At RT, addition of 25 to 40 μM TBA1 fully stabilized thrombin activityfor 3 months (FIG. 2A). At 37° C. less than 10% activity were lost after14 days at the presence of 25 μM TBA1 (FIG. 2B).

At 2-8° C., close to 100% thrombin activity were retained with 40 μMTBA1 after 180 days (FIG. 2C).

Remaining thrombin activities after incubation at 37° C. for 7 days inthe presence of different TBA1 concentrations as in FIG. 2B are plottedagainst the TBA1 concentrations (FIG. 2D). Thus, thrombin stabilizationby TBA1 is correlated with TBA1 concentration and therefore withthrombin inhibition. These data thus show that TBA1 stabilizes thrombinby virtue of inhibition of its autocatalytic activity.

Example 4: Physical Properties of Clot Prepared with Thrombin ContainingTBA and Antisense Oligonucleotide

The physical properties of a clot formed with thrombin containing TBAand antisense oligonucleotide were assessed. 8 IU/ml (about 0.08 μM)thrombin were incubated with 25 μM of TBA1 and 25 μM of antisenseoligonucleotide. After clot formation was initiated by mixing thethrombin with a 7% fibrinogen containing solution (BAC2), and 30 minutesincubation, elasticity of the resulting clot was tested.

The results (FIG. 3) show that the presence of TBA1 and the antisenseoligonucleotide does not change the elasticity of the clot, one of itsmost important physical properties.

Example 5: Effect of Antisense Oligonucleotide on the Rate of InhibitionReversibility

The rate at which the antisense oligonucleotide reverses the inhibitoryeffect of TBA1 was assessed. To this end, thrombin was diluted to 10IU/ml (˜0.1 μM) for the thrombin activity assay and TBA1 was added at 5,or 25 μM (resulting in 50:1 or 250:1 TBA1:thrombin ratio, respectively).All concentrations are final concentrations within the thrombin sample.The thrombin activity assay was initiated by adding a fibrinogencomponent including an equimolar amount of the antisenseoligonucleotides.

Four (4) volumes of fibrinogen solution containing AS were added to onevolume of thrombin solution, so that the final TBA1 concentration in the5 and 25 uM TBA1-containing thrombin solutions was, 1 and 5 μM,respectively, and thrombin was at 2 IU/ml (or ˜0.02 μM). TheTBA1/thrombin ratios remained unchanged. In order to assess potentialinhibitory effect of the antisense oligonucleotides, a controlcontaining antisense alone (without TBA1) was included.

Thrombin activity in this assay was measured as the time required forthrombin to clot the fibrinogen. Thus, 100% extrapolated activity wouldindicate that TBA1 inhibition was neutralized by the antisenseoligonucleotides at a time constant which is faster than the sensitivityof the assay ( 1/10^(th) of a second). Antisense oligonucleotides wereadded at a range between 1/10^(th) and 8-fold that of the TBA1, in orderto overcome the possibly too slow binding rate between the twocomponents. As can be seen in FIG. 4, at TBA1 final concentration 1 μM(4A) as well as, 5 μM (4B), there was a delay in the clotting reaction,even when the antisense oligonucleotide was added to the fibrinogensolution at large excess compared to TBA. Taken together, apparently theshort time available for TBA1 neutralization with the antisenseoligonucleotide when added in the fibrinogen component is not longenough to efficiently neutralize the TBA-1 mediated inhibition ofthrombin.

The molar ratio between TBA1:thrombin did also appear to have an effecton the reaction (50:1 or 250:1 TBA1:thrombin ratio resulted in recovery70% and 60%, respectively). While addition of an antisense excess of 5:1(antisense:TBA1) seemed to enhance the rate of TBA1 neutralization inthe reaction performed at lower TBA1 concentrations (5 μM antisense:˜MTBA1, FIG. 4A), a 4:1 or 8:1 excess did not elicit a higher recovery ofthrombin activity when the assay was performed at higher TBA1concentrations (20 and 40 μM antisense:5 μM TBA1, respectively, FIG.4B). Finally, antisense oligonucleotide added to thrombin without TBA1also caused a small but detectable reduction in the thrombin activity(FIGS. 4A and B).

Example 6: Incomplete Reversal of Large Excess TBA-Inhibited ThrombinActivity with Antisense Oligonucleotides

The data depicted in FIG. 1 showed that thrombin inhibition by TBA isfully reversible. Rapid reversal is a key attribute for any reversibleinhibitor to be used in a fibrin sealant mixture.

To test the time needed for complete reversal of thrombin inhibition byTBA1, we examined the actual time required for efficient reversal ofTBA1 added in excess. 25 μM of TBA1 were added to 10 IU/ml (0.1 μM) ofthrombin, a concentration that can be directly tested in the thrombinactivity assay. This represents a 250:1 TBA1:thrombin ratio. Antisenseoligonucleotide was added at an equimolar amount to TBA1 (25 μM) at time0, and samples were tested using the thrombin clotting assay at varioustime points thereafter (the thrombin:TBA1:antisense mixture was dilutedtogether in the reaction cuvette so that the molar ratio between themdoes not change). This assay was also performed for TBA2 with acorresponding antisense oligonucleotide. The results showed that whilethrombin activity measured after 60 seconds is approximately 7 IU/ml(aprox.70%), there was no improvement after 600 seconds, and activitydid not fully recover after 1800 seconds (FIG. 5). Countering TBA2 withantisense was even less efficient (FIG. 5). Surprisingly, a large excessof TBA over thrombin cannot be efficiently neutralized with antisenseoligonucleotide, even after long incubations.

Example 7: Drop Test with TBA1

Without wishing to be bound to theory, two factors may contribute to therate of the removal of inhibition by TBA1 from thrombin by antisenseoligonucleotide: the absolute concentration of the TBA, and the molarratio between TBA and thrombin. The effect of increasing theconcentration of thrombin, while maintaining TBA1 in the effectivestabilization range (˜5-25 μM), thus changing the molar ratio betweenthrombin and TBA1, was tested. The thrombin clotting assay cannot beperformed at thrombin concentrations used in fibrin sealants, asclotting is too rapid to be measured.

Therefore, the drop test assay, which is performed with 200 IU/mlthrombin (˜2 μM) was utilized. This assay is also, essentially, akinetic assay. Non-polymerized fibrinogen is liquid and slides down atilted slope, and the rate of fibrinogen polymerization induced bythrombin determines the time required for the mixture to clot and,consequently, to stop moving. This assay was performed in two ways.First, TBA1 was added to the thrombin component and antisenseoligonucleotides to the fibrinogen (BAC2) component. The interactiontime afforded to the two components is very short: from the moment thatboth mixtures are expressed from the common tip and until the mixture ispolymerized. Under these conditions, TBA1 was not rapidly enoughneutralized by the antisense oligonucleotide, and the distance traversedby the mixture was almost double that of control. Doubling the antisenseconcentration did not improve the rate of interaction (see FIG. 6,control without TBA1, left bar vs. 2^(nd) and 3^(rd) bar). In the secondset of experiments, TBA1 was added to the thrombin component, andsubsequently the antisense oligonucleotide was added and pre-incubatedfor several minutes (5-30). Here, the molar ratios between TBA1 andthrombin were critical. At the 12.5:1 molar ratio (25 μM TBA1:˜2 μMthrombin), there was a clear reduction of the distance travelled by theclot as compared to the previous test (FIG. 6, compare the 2^(nd) and3^(rd) bars to the 5^(th) bar). Surprisingly, upon reduction of theTBA1:thrombin molar ratio to that effectively used in the thrombinstabilization experiments (FIG. 2), 2.5:1 (5 μM TBA1:˜2 μM thrombin), nosignificant difference from control was observed (FIG. 6, 1 ^(st) vs.4^(th) bar).

These results demonstrated that the TBA1:thrombin ratio affects thereversibility of TBA1 inhibition by the antisense oligonucleotide.

In a modified drop test 1000 IU/ml thrombin, was effectively stabilizedby 25 μM TBA1 (FIG. 2). Rapid neutralization of TBA1 is a requirementfrom a fibrin sealant. To test this under real-life settings, the droptest assay was modified to include undiluted thrombin (1000 IU/ml) with25 μM TBA1. Antisense oligonucleotide was added to the thrombin/TBA1compartment just prior to the start of the test. This significantlydelayed clot formation (FIG. 7) as compared to control, uninhibitedthrombin. The same test was repeated, but with two minutes incubation ofthe antisense with thrombin/TBA1 prior to drop test start. Nosignificant differences were now seen compared to control (FIG. 7).

Taken together, when TBA1 was used with thrombin, at concentrationsefficiently stabilizing thrombin, antisense was efficient incounteracting thrombin inhibition, provided it was added to thrombin ˜2minutes prior to testing of the sealant, a time frame acceptable forpractical clinical use.

The drop test results, using actual drug product concentrations ofthrombin, and TBA1 concentrations shown to efficiently stabilizethrombin activity, indicated that the few minutes required to preparethe formulation for use are sufficient to allow effective neutralizationof TBA1 with antisense oligonucleotide.

Example 8: Preclinical Testing of Reversibly Stabilized Thrombin

To assess the use of the disclosed formulations in a preclinicalsetting, the capability of stabilized EVICEL® (a typical fibrin sealant)to stop bleeding in the heparinized rat kidney model was assessed(Macromol Biosci. 2010 Jan. 11; 10 (1):33-9. doi:10.1002/mabi.200900129. Hemostatic efficacy of biologicalself-assembling peptide nanofibers in a rat kidney model. Song H1, ZhangL, Zhao X.). In this model, a kidney was transected out of ananaesthetized rat and the main artery clamped. A transversal cut wasperformed through the entire cross-section of the kidney and fibrinsealant applied. The clamp was then released and the efficiency of thehemostatic composition assessed by the amount of blood lost through thekidney surface. In order to prevent endogenous hemostasis, the rats werepre-injected with 300 IU/kg body weight of heparin. EVICEL® (FIG. 8,control) vs. EVICEL® in which the thrombin component was inhibited with25 μM of TBA1, and then contacted with the same amount of antisenseoligonucleotide just prior to application (FIG. 8, test) were tested.The time elapsed between applying the antisense oligonucleotides and theactual spraying of the fibrin sealant (the time required to assemble theEVICEL® two component double barreled syringe, and spray tip) isapproximately 2 minutes. As can be seen in FIG. 8, the test group was atleast as efficient as the control group in achieving hemostasis.

Example 9: Reversal of TBA-Inhibited Thrombin Activity withStreptavidin-Sepharose-Immobilized Biotinylated AntisenseOligonucleotides

To examine reversal of the thrombin inhibitory effect of TBA1 byimmobilized antisense oligonucleotides, a 5′ biotinylated (Btn)derivative of antisense TBA1 (AS) was synthesized ([Btn]AS).Streptavidin-Sepharose resin was prepared according to manufacturerinstructions (GE product code 17-5113-01; having a binding capacitywhich is biotin >300 nmol/ml medium). Briefly, 100 nmol [Btn]AS was setto bind to 1 ml settled Streptavidin-Sepharose beads (Mean particle size34 μm), representing a minimum of 3 fold excess of streptavidin tobiotin over the [Btn]AS, for 15 minutes at ambient temperature. Bindingreaction was carried out according to manufacturer instructions inreaction buffer: 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5. A complexSepharose-Streptavidin/[Btn]AS was formed (Beads-[Btn]AS). The complexwas sedimented by 1 min spin down at approximately 5000 g, supernatantdecanted. Washing was carried out three times (in reaction buffer). TheBeads-[Btn]AS complex was resuspended with reaction buffer to 5% (v/v)slurry.

To establish a base-line for the experiment, 10 μl of 1000 IU/mlThrombin-were mixed with 0, 0.1, 0.2, 0.5 and 1 μM TBA1 in a finalvolume of 1 ml. The mixture was incubated for 30 min at ambienttemperature and thrombin activity was determined to be 10.99 and 2.38IU/ml with 0 or 1 μM TBA1, respectively (FIG. 9A). To determine theextent of the reversal inhibition, equimolar amounts of antisense in{[Btn]AS} or {Beads− [Btn]AS} were added to pre-incubated base-linemixtures of thrombin and TBA1, thrombin activity was assayed after 15min incubation at ambient temperature. Reversal of inhibition of TBA1 by{[Btn]AS} or {Beads− [Btn]AS} was equal and incomplete (FIG.9A)—thrombin activity increased from 6.71 IU/ml (without AS) to 9.081U/ml or 8.841 U/ml for 0.1 μM TBA1/AS instead of 10.71 IU/ml withoutTBA1 (FIG. 1) and increased from 2.38 IU/ml (without AS) to 5.9 IU/ml or5.76 IU/ml for 1 μM TBAVAS instead of 10.26 IU/ml without TBA1 (FIG. 1).FIG. 1 was used as a reference for the inhibition level of TBA1+AS.

Without being bound by the mechanism, complete reversal of inhibitionwas not obtained, most likely because of steric hindrance. Addition of alinker sequence may allow optimal binding of antisense nucleotide toTBA1.

To test possible artifacts of beads alone on thrombin activity, 200microliter of beads (5% slurry) and 10 μl of thrombin 1000 IU/ml weremixed with thrombin dilution buffer (0.4% sodium citrate di-hydrate,0.9% NaCl, 1% BSA) to a final volume of 1 ml. Thrombin activity assayedafter 30 min incubation at ambient temperature was 10.99 IU/ml.

To test possible artifacts of [Btn]AS on thrombin activity, 50microliter of [Btn]AS (20 μM) and 10 μl of thrombin 1000 IU/ml weremixed with thrombin dilution buffer to a final volume of 1 ml. Thrombinactivity assayed after 30 min incubation at ambient temperature was10.58 IU/ml.

To test possible artifacts of Beads− [Btn]AS on thrombin activity, 200microliter of Beads− [Btn]AS (5% slurry) and 10 μl of thrombin 1000IU/ml were mixed with thrombin dilution buffer to a final volume of 1ml. Thrombin activity assayed after 30 min incubation at ambienttemperature was 10.34 IU/ml.

The results show that beads, Beads− [Btn]AS and [Btn]AS do not affectthrombin activity.

To establish kinetics of reversal of inhibitory effect of TBA1 equimolaramounts of Beads− [Btn]AS were added to pre-incubated base-line mixturesafter 2, 5, 10 and 15 min. The mixtures were tested immediately, afterthe addition of AS, for thrombin activity. Maximal reversal ofinhibition was observed after 10 and 15 min for 0.1 or 0.2 and 0.5 or 1μM TBA1, respectively (FIG. 9B). Thrombin inhibition and re-activationtiming can be optimized as needed, depending on the intended use.

The data gathered from the above described experiments show thatthrombin binding oligonucleotides (e.g. aptamers) function as efficientstabilizers of highly concentrated, purified thrombin in liquidformulation. Their concentration preferably falls within a defined rangeof molar ratios compared to the thrombin. The reversal of TBAs ispossible and efficient, provided that the molar ratio of TBA:thrombindoes not exceed 10:1, and is preferably about 2.5:1 to about 4:1. Theamount of antisense oligonucleotides used to counter the TBA preferablydoes not exceed the amount of TBA by more than ˜20% so that freeantisense oligonucleotide concentration does not exceed ˜5 μM which isshown here to also inhibit thrombin activity on fibrinogen in a clottingassay. Under these conditions, the time required for efficientcountering TBA is <2 minutes, which is approximately the time requiredin the clinic/surgery to assemble and position the device forapplication. Thus, this invention represents an applicable means toefficiently stabilize highly purified thrombin in the liquidformulation, in a reversible manner, with no expected toxicity orimmunogenicity.

Although various embodiments have been described herein, manymodifications and variations to those embodiments may be implemented.Also, where materials are disclosed for certain components, othermaterials may be used. The foregoing description and following claimsare intended to cover all such modification and variations.

The disclosure of applications, patents and publications, cited above ishereby incorporated by reference.

Any patent, publication, or other disclosure material, in whole or inpart, that is said to be incorporated by reference herein isincorporated herein only to the extent that the incorporated materialsdoes not conflict with existing definitions, statements, or otherdisclosure material set forth in this disclosure. As such, and to theextent necessary, the disclosure as explicitly set forth hereinsupersedes any conflicting material incorporated herein by reference.

Citation or identification of any reference in this application shallnot be construed as an admission that such reference is available asprior art to the invention.

Section headings are used herein to ease understanding of thespecification and should not be construed as necessarily limiting.

1-33. (canceled)
 34. A delivery applicator comprising: a barrel holdingan oligonucleotide inhibited thrombin; and a vessel having a deliveryopening and holding an antisense oligonucleotide; wherein the barrel andthe vessel are capable of being in fluid communication, so that afterfluid communication and contact of the inhibited thrombin with theantisense, thrombin activity is increased before delivering thrombinthrough the delivery opening.
 35. The delivery applicator of claim 34,wherein the fluid communication is via a re-sealable opening positionedbetween the barrel and the vessel.
 36. The delivery applicator of claim34, further comprising a barrel having a delivery opening and holdingfibrinogen.
 37. The delivery applicator of claim 34, wherein theantisense is bound to a solid phase.
 38. A delivery applicatorcomprising: a container holding an oligonucleotide inhibited thrombin;and an antisense oligonucleotide, wherein the oligonucleotide inhibitedthrombin and the antisense oligonucleotide are held in separate chambersthat are capable of being in fluid communication, so that allowing fluidcommunication between the chambers and contact between the inhibitedthrombin and the antisense results in increased thrombin activity beforedelivery, and wherein the container has an opening for deliverytherethrough of the thrombin.
 39. The delivery applicator of claim 38,wherein the antisense is bound to a solid phase.
 40. (canceled)